ABSTRACT (PROJECT SUMMARY) Genome wide association studies (GWAS) of late-onset AD have identified more than 20 genes to be associated with disease risk, with variation in the apolipoprotein E (APOE) gene being the strongest risk factor. APOE is a lipoprotein that is known to have functions in cholesterol metabolism, but the mechanisms by which it imparts AD risk remain poorly understood. APOE has three isoforms ?2 (E2) , ?3 (E3) , and ?4 (E4) where individuals with the ?4 allele are 3-12 times more likely to develop AD and those with ?2 allele have reduced risk of developing AD. APOE4 affects amyloid beta (A?) aggregation, metabolism, and plaque load in both individuals with AD and mouse models of AD. APOE4 also differentially affects microglial-mediated inflammatory responses in the brain as well as endothelial-mediated clearance of A? in the cerebrovasculature, suggesting that APOE-associated AD risk may in part be driven by dysfunctional inflammatory and vascular responses to A? pathology. At present, there is a significant gap in our knowledge of what molecular constituents mediate microglial and/or endothelial disease mechanisms in APOE-linked AD. The consideration of these mechanisms can aid our understanding of proteins that may work together to affect the disease state or protection, create a framework for the pathophysiology of the disease, and guide exploration into therapeutic targets. The long-term goal of this research is to better understand how APOE genotype effects cellular phenotypes in brain. To achieve this goal, I will use data-driven proteomic and systems biology approaches to integrate human and mouse model studies and resolve the impact of APOE genotype on AD risk. Specifically, the experiments outlined in this proposal will test the central hypothesis that microglial and endothelial cellular phenotypes in AD are caused by APOE genotype. This hypothesis is supported by my exciting preliminary data that show APOE genotype impacts global proteomic alterations, microglial and endothelial cell abundance, and cell-type profiles in human AD brains. These proteomic profiles imply a linkage between the microglia, endothelia, and APOE-mediated pathophysiologies in AD brain, but since they were obtained from whole brain tissue, the cell-type specific contributions to the proteomic alterations may be masked. Given this, we have recently optimized cell isolation and mass-spectrometry (MS)-based techniques to comprehensively characterize, for the first time, proteomic profiles of brain endothelial cells in parallel with microglia from mouse brain. I will characterize the proteomic profile of isolated cells from the brains of EFAD transgenic mice to unravel the impact of APOE genotype on microglial and endothelial molecular phenotypes as well as characterize age- related changes in these cells. Importantly, these data will provide insight into convergent APOE genotype driven mechanisms in microglial and endothelial cells in both mice and human brain.